Photochemotherapeutic compositions

ABSTRACT

The invention provides pharmaceutical compositions comprising a protoporphyrin precursor photochemotherapeutic agent together with vascular stroma-localizing photosensitizers, optionally together with at least one surface penetration assisting agent and optionally with one or more chelating agents, and use of the same in treating disorders or abnormalities which are responsive to PDT, preferably exhibiting synergistically enhanced therapy, kits comprising same and methods of therapy and diagnosis.

[0001] The present invention relates to pharmaceutical compositions foruse in the treatment of disorders or abnormalities of the skin and otherbody surfaces by photochemotherapy.

[0002] Abnormalities or disorders, such as neoplasms or psoriaticplaques, of the skin or other epithelial, e.g. mucosal, organs areconventionally treated by surgery, radiotherapy, cryotherapy orchemotherapy. These treatments however, often have significant andserious drawbacks such as toxicity, carcinogenicity, or other unpleasantside effects or general discomfort resulting from the treatment.

[0003] Photochemotherapy or photodynamic therapy (PDT) as it is alsoknown, is a recently up-coming technique for the treatment of variousabnormalities or disorders of the skin or other epithelial organs,especially cancers or pre-cancerous lesions, as well as certainnon-malignant lesions for example skin complaints such as psoriasis.Photochemotherapy involves the application of photosensitizing(photochemotherapeutic) agents to the affected area of the body orsystemic application, followed by exposure to photoactivating light inorder to activate the photosensitizing agents and convert them intocytotoxic form, whereby the affected cells are killed or theirproliferative potential diminished.

[0004] A range of photosensitizing agents are known, including notablythe psoralens, the porphyrins, the chlorins and the phthalocyanins. Suchdrugs become toxic when exposed to light.

[0005] Photosensitizing drugs may exert their effects by a variety ofmechanisms, directly or indirectly. Thus for example, certainphotosensitizers become directly toxic when activated by light, whereasothers act to generate toxic species, e.g. oxidising agents such assinglet oxygen or other oxygen-derived free radicals, which areextremely destructive to cellular material and biomolecules such aslipids, proteins and nucleic acids. Psoralens are an example of directlyacting photosensitizers; upon exposure to light they form adducts andcross-links between the two strands of DNA molecules, thereby inhibitingDNA synthesis. The unfortunate risk with this therapy is that unwantedmutagenic and carcinogenic side effects may occur.

[0006] This disadvantage may be avoided by selecting photosensitizerswith an alternative, indirect mode of action. For example porphyrins,which act indirectly by generation of toxic oxygen species, have nomutagenic side effects and represent more favourable candidates forphotochemotherapy. Porphyrins are naturally occurring precursors in thesynthesis of heme. In particular, heme is produced when iron (Fe³⁺) isincorporated in protoporphyrin IX (Pp) by the action of the enzymeferrochelatase. Pp is an extremely potent photosensitizer, whereas hemehas no photosensitizing effect.

[0007] One such porphyrin-based drug, Photofrin® (Gomer and Dougherty,Cancer Research, 39, p146-151, 1979; originally named Photofrin II) hasrecently been approved as a photosensitizer in the therapy of certaincancers. Photofrin® consists of large oligomers of porphyrin and it doesnot readily penetrate the skin when applied topically and must thereforebe administered systemically. Thus, its main disadvantage is that sinceit must be administered parenterally, generally intravenously, it causesphotosensitization of the skin which may last for several weeksfollowing i.v. injection. Similar problems exist with otherporphyrin-based photosensitizers such as the so-called “hematoporphyrinderivative” (Hpd) (Lipson et al., J. Natl. Cancer Ins., 60, p1-10, 1961)which has also been reported for use in cancer photochemotherapy (seefor example S. Dougherty., J. Natl. Cancer Ins., 52, p1333, 1974; Kellyand Snell, J. Urol., 115, p150, 1976). Hpd is a complex mixture obtainedby treating haematoporphyrin with acetic and sulphuric acids, afterwhich the acetylated product is dissolved with alkali. Clearly, thereare disadvantages in using an undefined mixture as a drug. Moreoversince Hpd must also be administered by injection, it suffers from thesame type of undesirable photosensitization drawback as does Photofrin®.

[0008] To overcome these problems, precursors of Pp have beeninvestigated for photochemotherapeutic potential. In particular the Ppprecursor 5-aminolevulinic acid (ALA) has been investigated as aphotochemotherapeutic agent for certain skin cancers. ALA, which isformed from succinyl CoA and glycine in the first step of hemesynthesis, is to a limited extent able to penetrate the skin and lead toa localised build-up of Pp; since the action of ferrochelatase (themetallating enzyme) is the rate limiting step in heme synthesis, anexcess of ALA leads to accumulation of Pp, the photosensitizing agent.Thus, by applying ALA topically to skin tumours, and then after severalhours exposing the tumours to light, a beneficial photochemotherapeuticeffect may be obtained (see for example WO91/01727). Since the skincovering basaliomas and squamous cell carcinomas is more readilypenetrated by ALA than healthy skin, and since the concentration offerrochelatase is low in skin tumours, it has been found that topicalapplication of ALA leads to a selectively enhanced production of Pp intumours.

[0009] However, whilst the use of ALA represents a significant advancein the art, photochemotherapy with ALA is not always entirelysatisfactory. ALA is not able to penetrate all tumours and other tissueswith sufficient efficacy to enable treatment of a wide range of tumoursor other conditions and ALA also tends to be unstable in pharmaceuticalformulations. Some of these problems may be overcome by using ALAderivatives, for example ester derivatives such as ALA-methylester,ALA-ethylester, ALA-propylester, ALA-hexylester, ALA-heptylester andALA-octylester and salts thereof as described in our co-pendingapplication WO96/28412.

[0010] Like ALA, the ester derivatives exert their effects by enhancingproduction of Pp; upon delivery to the desired site of action hydrolyticenzymes such as esterases present in the target cells break down theesters into the parent ALA, which then enters the haem synthesis pathwayand leads to a build-up of Pp. However, the ester derivatives have anumber of advantages over ALA itself. Firstly, they are more lipophilicand better able to penetrate skin and other tissues as compared withALA; the penetration is both deeper and faster. This is an importantadvantage, especially for topical administration. Secondly, the estersare better enhancers of Pp production than ALA; Pp production levelsfollowing administration of the ALA esters are higher than with ALAalone. Thirdly, the ALA esters demonstrate improved selectivity for thetarget tissue to be treated, namely the Pp production-enhancing effectis localised to the desired target lesion and does not spread to thesurrounding tissues. This is especially evident with tumours. Finally,the esters appear to localise better to the target tissue uponadministration. This may be especially important for systemicapplication, since it means that undesirable photosensitization effects,as reported in the literature for other porphyrin-basedphotosensitizers, may be reduced or avoided.

[0011] Whilst such ALA esters represent a considerable advance in thefield of photochemotherapy, not all abnormalities or disorders respondto PDT using known methods to prevent tumour growth and thus there isstill a need for better and alternative photochemotherapeutic agents toretard or prevent tumour growth. The present invention thus aims toprovide photochemotherapeutic compositions which have an enhancedphotochemotherapeutic effect over those described in the prior art.

[0012] Studies conducted by the authors have shown that efficienteradication of tumours by PDT requires destruction of both cellularcomponents and also vascular stroma of tumours (Peng & Moan, Br. J.Cancer, 72, p565-574, 1995; Peng et al., Cancer Res., 55, p2620-2626,1995 and Peng et al., Ultrastructural Pathology, 20, p109-129, 1996).ALA has proven utility in treating tumours and the PpIX synthesizedendogenously from ALA localizes within tumour cells. Furthermore,locally applied ALA does not cause skin sensitization and has nomutagenic effect on the DNA of cells. Systemically applied ALA shows nosensitization 24 hours after administration. As mentioned previouslyhowever, ALA is not able to penetrate all tumours and has only beenfound to have good efficacy for the treatment of superficial lesions ofthe skin with a thickness less than 2-3 mm. No good clinical resultshave been obtained using topically or systemically administered ALA-PDTon thicker skin lesions or thicker lesions of the aerodigestive tract orother internal hollow organs. Photofrin® is known to distribute mainlyin vascular stroma of tumours, but as mentioned above, is associatedwith a prolonged risk of skin photosensitization.

[0013] However, it has now surprisingly been found, that the use of avascular stroma-localizing photosensitizer, e.g. Photofrin®,tetra(meso-hydroxyphenyl)chlorin (m-THPC), chlorin e6; aluminiumphthalocyanine di-sulfonate or aluminium phthalocyanine tetra-sulfonatein combination with a protoporphyrin precursor photochemotherapeuticagent, e.g. ALA or its methyl or butyl esters, enhances the efficiencyof PDT relative to the use of one of the agents alone. A synergisticeffect was observed between the vascular stroma-localizingphotosensitizer and the protoporphyrin precursor photochemotherapeuticagent, resulting in improved suppression of tumour growth compared tothe expected additive effect of the agents alone. This advantageous,synergistic effect was surprisingly observed even when using thevascular stroma-localizing agent at a less than therapeutic dose(sub-therapeutic) which whilst not effective at reducing tumour growth,reduces or avoids the risk of skin photosensitivity. For example, thegrowth of tumours treated in this way were found to be reduced by usingALA at a therapeutic dose and Photofrin® (or m-THPC) at a lownon-therapeutic level. The reduction in growth was significantly greaterwhen compared to the additive effects of results obtained using ALA at atherapeutic dose or Photofrin® (or m-THPC) at a therapeutic dose. Thissuggests a hitherto unrecognized synergistic effect between thesedifferent types of photochemotherapeutic agents, even at non-therapeuticdoses.

[0014] The synergistic effect, even at sub-therapeutic levels, hassignificant clinical implications. Firstly, improved PDT is achievedwhich is not limited to superficial skin lesions, but may also be usedto treat thick skin lesions and superficial lesions of internal holloworgans, and secondly, if sub-therapeutic doses of the vascularstroma-localizing photosensitizer are employed, the skin phototoxicityassociated with these agents may be avoided.

[0015] In one aspect, the present invention thus provides apharmaceutical composition for the treatment of disorders orabnormalities of external or internal surfaces of the body which areresponsive to photochemotherapy, comprising a protoporphyrin precursorphotochemotherapeutic agent together with a vascular stroma-localizingphotosensitizer, optionally together with at least one surfacepenetration assisting agent and optionally with one or more chelatingagents. In particular, the therapeutic efficacy of thephotochemotherapeutic agents is enhanced, ie. PDT is enhanced relativeto the use of one of the agents alone. More particularly, thetherapeutic efficacy is synergistically enhanced. In a preferred aspectof the invention, the vascular stroma-localizing photosensitizer isprovided at a sub-therapeutic dose.

[0016] Alternatively viewed, the invention can be seen to provide theuse of a protoporphyrin precursor photochemotherapeutic agent togetherwith a vascular stroma-localizing photosensitizer, optionally togetherwith at least one surface penetration assisting agent and optionallywith one or more chelating agents in the preparation of a compositionfor the treatment of disorders or abnormalities of external or internalsurfaces of the body which are responsive to photochemotherapy.

[0017] The invention also extends to novel compositions ofprotoporphyrin precursor photochemotherapeutic agents and vascularstroma-localizing photosensitizers, optionally together with at leastone surface penetration assisting agent and optionally with one or morechelating agents.

[0018] It will be appreciated that certain vascular stroma-localizingphotosensitizers, e.g Photofrin®, can not be administered topically, andthus unless both photochemotherapeutic agents of compositions of theinvention are administered parenterally, the administration will be byuse of separate preparations either administered at the same time orfollowing one another.

[0019] Thus, viewed from a further aspect, the invention thus provides aproduct comprising a protoporphyrin precursor photochemotherapeuticagent and a vascular stroma-localizing photosensitizer, optionallytogether with at least one surface-penetration assisting agent, andoptionally one or more chelating agents as a combined preparation forsimultaneous, separate or sequential use in treating disorders orabnormalities of external or internal surfaces of the body which areresponsive to photochemotherapy.

[0020] Furthermore, the use of a protoporphyrin precursorphotochemotherapeutic agent and a vascular stroma-localizingphotosensitizer, optionally together with at least onesurface-penetration assisting agent, and optionally one or morechelating agents in the preparation of a product for simultaneous,separate or sequential use in treatment of disorders or abnormalities ofexternal or internal surfaces of the body which are responsive tophotochemotherapy, forms a further aspect of the invention.

[0021] As used herein, “protoporphyrin precursor photochemotherapeuticagents” refers to structural precursors of protoporphyrin andderivatives thereof which function as photochemotherapeutic agents, forexample ALA, porphobilinogen or precursors or derivatives thereof, whichform a preferred aspect of the invention. Generally such agents localizeto cells of the lesion, e.g. a tumour or diseased cell.

[0022] “Vascular stroma-localizing agents” refers to agents whichgenerally localize to the vascular stroma after administration. Suitablevascular stroma-localizing agents include:

[0023] HpD;

[0024] Hematoporphyrines such as Photofrin® (Quadra Logic TechnologiesInc., Vancouver, Canada) and Hematoporphyrin IX (HpIX);

[0025] Photosan III (Seehof Laboratorium GmbH, Seehof,Wesselburenerkoog, Germany);

[0026] Clorins such as tetra(m-hydroxyphenyl)chlorins (m-THPC) and theirbacteriochlorins (Scotia Pharmaceuticals Ltd, Surrey, UK),mono-L-aspartyl chlorin e6 (NPeG) (Nippon Petrochemical Co., CA, USA),chlorin e6 (Porphyrin Products Inc.), benzoporphyrins (Quadra LogicTechnologies Inc., Vancouver, Canada)(e.g. benzoporphyrin derivativemonoacid ring A, BPD-MA) and purpurines (PDT Pharmaceuticals Inc., CA,USA) (e.g. tin-ethyl etiopurpurin, SnET2);

[0027] phthalocyanines (e.g. zinc-(Quadra Logic Technologies Inc.,Vancouver, Canada), some aluminium- or silicon phthalocyanines, whichmay be sulfonated, in particular sulfonated phthalocyanines such asaluminium phthalocyanine di-sulfonate (A1PcS_(2a)) or aluminiumphthalocyanine tetra-sulfonate (A1PcS₄));

[0028] porphycenes;

[0029] hypocrellins;

[0030] Protoporphyrin IX (PpIX);

[0031] Hematoporphyrin di-ethers;

[0032] Uroporphyrins;

[0033] Coproporphyrins;

[0034] Deuteroporphyrin; and

[0035] Polyhematoporphyrin (PHP), and precursors and derivativesthereof.

[0036] As mentioned previously, Photofrin® comprises a mixture ofdifferent components and each of these separate components orcombinations thereof may be used to provide the vascularstroma-localizing agent.

[0037] “Vascular stroma” is intended to signify the vascular connectivetissue, matrix and its components and nerves in addition to cells suchas macrophages and fibroblasts present in the vascular system and othercells which infiltrate into the stroma. It will be appreciated that theregion of localization will depend on the time post-administration atwhich localization is determined. Thus, photosensitizers which initiallylocalize in cells may relocate to the stroma, and vice versa. Forexample, aluminium phthalocyanine di-sulfonate localizes initially tothe stroma whereas 24-72 hours post-injection the majority of the agentis found in cells.

[0038] In general however, vascular stroma-localizing agents areconsidered to be those present in the stroma in the 24 hours followingadministration. This may however be manipulated by performing PDT atdifferent times post-administration of the agent such that the agent(s)behaves appropriately as a vascular stroma or lesion-localizing agent atthe time of irradiation.

[0039] Preferably the protoporphyrin precursor is ALA or a precursor orderivative thereof and the vascular stroma-localizing photosensitizer isa Hematoporphyrin (particularly Photofrin®), a chlorin (particularlym-THPC or chlorin e6) or a sulphonated phthalocyanine (particularlyaluminium phthalocyanine di-sulfonate or aluminium phthalocyaninetetra-sulfonate).

[0040] The term “precursors” as used herein refers to precursors for theagent which are converted metabolically to that agent and are thusessentially equivalent to that agent, e.g. ALA. Thus the term“precursor” covers biological precursors for protoporphyrin in themetabolic pathway for haem biosynthesis. “Derivatives” includepharmaceutically acceptable salts and chemically modified agents, forexample esters such as ALA esters as described hereinbefore.

[0041] Surface-penetration assisting agents may be used which have abeneficial effect in enhancing the photochemotherapeutic effect. Suchagents may be used even when the photochemotherapeutic agents are notadministered topically. Dialkylsulphoxides such as dimethylsulphoxide(DMSO) are especially preferred. This is described in detail in WO95/07077.

[0042] The surface-penetration assisting agent may be any of theskin-penetration assisting agents described in the pharmaceuticalliterature e.g. HPE-101 (available from Hisamitsu), DMSO and otherdialkylsulphoxides, in particular n-decylmethyl-sulphoxide (NDMS),dimethylsulphacetamide, dimethylformamide (DMFA), dimethylacetamide,glycols, various pyrrolidone derivatives (Woodford et al., J. Toxicol.Cut. & Ocular Toxicology, 5, p167-177, 1986), and Azone® (Stoughton etal., Drug Dpv. Ind. Pharm., 9, p725-744, 1983), or mixtures thereof.

[0043] DMSO however has a number of beneficial effects and is stronglypreferred. Thus, in addition to the surface-penetration assisting effect(DMSO is particularly effective in enhancing the depth of penetration ofthe active agent into the tissue), DMSO has anti-histamine andanti-inflammatory activities, leading to a reduction in pain during thelight exposure process. In addition, DMSO has been found to increase theactivity of the enzymes ALA-synthase and ALA-dehydrogenase (the enzymeswhich, respectively, form and condense ALA to porphobilinogen) therebyenhancing the formation of the active form, Pp.

[0044] However, in certain conditions such as psoriasis, the lesions arerelatively easily penetrated and the penetrating agent may be lessbeneficial. In some circumstances, for example in the case of skincancers where the lesions are difficult to penetrate, the surfacepenetration assisting agent may be applied in a preliminary step,generally at a higher concentration.

[0045] Thus, the various active components need not be appliedsimultaneously within the same composition, but may, according toclinical need, be administered separately and sequentially. Indeed, ithas been observed that in many cases a particularly beneficialphotochemotherapeutic effect may be obtained by pre-treatment with thesurface-penetration assisting agent in a separate step, prior toadministration of the photochemotherapeutic agents. Furthermore, in somesituations a pre-treatment with the surface-penetration assisting agent,followed by administration of the photochemotherapeutic agent inconjunction with the surface-penetration assisting agent has been foundto be beneficial. When a surface-penetration assisting agent is used inpre-treatment this may be used at high concentrations, e.g. up to 100%(w/w). If such a pre-treatment step is employed, thephotochemotherapeutic agent may subsequently be administered up toseveral hours following pre-treatment eg. at an interval of 5-60 minutesfollowing pre-treatment.

[0046] Malik et al in Proceedings of Photodynamic Therapy of Cancer,2078, p355-362, 1993, described in vitro studies of the effects of ALA,on induction of protoporphyrin biosynthesis, and subsequent killing byphotodestruction, of B16 melanoma cells in culture, which had previouslybeen incubated with DMSO as differentiation inducer and/orallyl-isopropyl-acetamide as porphyrogenic agent, to increase endogenousporphyrin levels prior to incubation with the ALA.

[0047] Doodstar et al in Biochemical Pharmacology, 42(6), p1307-1303,1991, describe an investigation into the effects of culture conditionson hepatocytes in culture, and in particular the effects of ALA andDMSO, alone or in combination, on increasing the activities ofcytochrome P450-dependent mixed function oxidase and UDP-glucuronosyltransferase, by increasing intracellular haem concentrations, inhepatocyte cells in culture.

[0048] Chelating agents are optionally contained in the pharmaceuticalcomposition or product of the invention. Such agents may be useful fortwo effects, firstly to enhance the stability of the protoporphyrinprecursor photochemotherapeutic agent, e.g. ALA and secondly to enhanceaccumulation of Pp. The latter effect is achieved by the chelation ofiron, thereby preventing the inactivating action of the enzymeferrochelatase in incorporating the metal into Pp, leading to Ppbuild-up. The photosensitizing effect is thus enhanced.

[0049] Hanania et al in Cancer Letters, 65, p127-131, 1992 propose theuse of ALA in combination with-chelating agents in photochemotherapy oftopically treated tumours.

[0050] Aminopolycarboxylic acid chelating agents are particularlysuitable for use in this regard, including any of the chelants describedin the literature for metal detoxification or for the chelation ofparamagnetic metal ions in magnetic resonance imaging contrast agents.Particular mention may be made of EDTA, CDTA (cyclohexane diaminetetraacetic acid), DTPA, DOTA and 1,10-phenanthroline. EDTA ispreferred, especially for the stabilisation of ALA. To achieve theiron-chelating effect, desferrioxamine and other siderophores may alsobe used, e.g. in conjunction with aminopolycarboxylic acid chelatingagents such as EDTA.

[0051] The compositions of the invention or used according to theinvention may additionally be formulated and/or administered with otheragents, to improve the efficacy of PDT. Thus for example, angiogenesisinhibitors (anti-angiogenic drugs) which have been found to be usefulfor treating tumours (O'Reilly et al., Nature Medicine, 2, p689-692,1996; Yamamoto et al., Anticancer Research, 14, p1-4, 1994; and Brookset al., J. Clin. Invest., 96, p1815-1822, 1995) may be used togetherwith compositions of the invention in PDT to further damage the vascularsystem of the tumour. Angiogenesis inhibitors which may be used includeTNP-470 (AGM-1470, a synthetic analogue of a fungal secretion productcalled fumagillin; Takeda Chemical Industries Ltd., Osaka, Japan),angiostatin (Surgical Research Lab. at Children's Hospital MedicalCenter of Harvard Medical School) and integrin α_(v)β₃ antagonists (e.g.monoclonal antibody to intefrin α_(v)β₃, The Scripps Research Institute,LaJolla, Calif.).

[0052] Alternatively, or additionally, immunotherapy agents (e.g.antibodies or effectors such as macrophage activating factor) orchemotherapy agents may be used to improve PDT according to theinvention. Administration of these supplementary agents should beperformed in terms of route, concentration and formulation, according toknown methods for using these agents. These additional agents may beadministered before, after or during PDT, depending on their function.For example, angiogenesis inhibitors may be added 5-10 days after PDT toprevent tumour regrowth.

[0053] Glucose has also been found to assist PDT when applied eithertopically or systemically. Although not wishing to be bound by theory,it appears that administration of glucose results in a lowering of pHwhich increases the hydrophobic properties of protoporphyrins such thatthey can penetrate cells more easily. When topical administration iscontemplated, conveniently the formulation, e.g. a cream, may contain0.01 to 10% glucose (w/w).

[0054] A preferred composition or product according to the invention,comprises ALA or a precursor or derivative thereof, Photofrin®, DMSO,EDTA and desferrioxamine.

[0055] As mentioned above, a synergistic effect has been observed,between the protoporphyrin precursor and the vascular stroma-localisingphotochemotherapeutic agent, whereby the efficiency of PDT is enhanced.Thus, this enables sub-therapeutic dosages of the photochemotherapeuticagent to be used ie. dosages which, were the individualphotochemotherapeutic-agent to be administered on its own, would notsuffice-to achieve a beneficial photochemotherapeutic effect.

[0056] It has in particular been found that beneficial results may beobtained using the protoporphyrin precursor agent, preferably ALA or aderivative thereof, at a therapeutic dose range, standard for PDT usingsuch a photochemotherapeutic agent solely, in conjunction with asub-therapeutic dose of the vascular stroma-localising agent, preferablyPhotofrin®.

[0057] The concentration of the protoporphyrin precursorphotochemotherapeutic agent, e.g. ALA in the composition is convenientlyin the range 1 to 40%, e.g. 2 to 25, preferably 5 to 20%; theconcentration of the vascular stroma-localizing photosensitizer, e.g.Photofrin® is conveniently in the range 0.1 to 1% or m-THPC isconveniently in the range 0.01-10%, the concentration of chelating agentis preferably in the range 1 to 20% e.g. about 2 to 10%, e.g. 2.5%; theconcentration of surface penetration assisting agent, e.g. DMSO, ispreferably in the range 2 to 50% e.g. about 10%. All percentages statedabove are by weight. The concentration of agent which is requireddepends on the particular agent which is used and clearly should bemodified as appropriate according to information and techniques known inthe art. Furthermore, it will be appreciated that the concentration useddepends on the method of application and on the time for which thecomposition is applied. However, as mentioned above, where the surfacepenetration assisting agent is administered separately in a preliminarystep, it may be applied at higher concentrations, even up to 100%.Compositions of the invention may be administered exclusively topically(by application to internal or external surfaces using for example acream, instillation, local internal administration/injection orinhalation) or systemically (e.g. orally or by intravenous injection) orby a combination of these methods in which one or more components of thecomposition is administered topically and the other components areadministered systemically.

[0058] The total dosage of the vascular stroma-localizingphotosensitizer administered, e.g. by intravenous administration ispreferably in the range of 0.01 to 10 mg/kg body weight, for example forPhotofrin® preferably 0.01 to 1 mg/kg body weight (sub-therapeutic dose)or for m-THPC preferably 0.01 to 0.2 mg/kg body weight and for theprotoporphyrin precursor photochemotherapeutic agent in the range of 1to 500 mg/kg, e.g. 1 to 250 mg/kg, for example for ALA in the range 1 to250 mg/kg, preferably 20 to 70 mg/kg body weight.

[0059] It will be appreciated that the dosage required depends on themode and route of administration, the agent employed and the lesion tobe treated. Whilst sub-therapeutic doses of the vascularstroma-localizing photochemotherapeutic agent are preferred, this may beincreased if for example a large thick lesion or a difficult type ofdisease (e.g. melanoma) is to be treated. The observed synergisticeffect allows the levels of both the vascular stroma-localizing agentand the protoporphyrin precursor agent to be reduced below normaltherapeutic levels.

[0060] Alternatively viewed, this aspect of the invention also providesa kit for use in photochemotherapy of disorders or abnormalities ofexternal or internal surfaces of the body comprising:

[0061] a) a first container containing a protoporphyrin precursorphotochemotherapeutic agent, e.g. ALA or a precursor or derivativethereof;

[0062] b) a second container containing a vascular stroma-localizingphotosensitizer, e.g. Photofrin® or m-THPC; and optionally

[0063] c) at least one surface-penetrating agent contained within saidfirst or second container or in a third container; and/or

[0064] d) one or more chelating agents contained either within saidfirst, second or third container or in a fourth container;

[0065] wherein said first or second container may be absent and theagent or photosensitizer of a) or b) above is present in one of theother containers present in the kit.

[0066] Additional components of the kit may also be provided such asangiogenesis inhibitors or glucose as mentioned hereinbefore.

[0067] The abnormalities and disorders which may be treated according tothe present invention include any malignant, pre-malignant andnon-malignant abnormalities or disorders responsive to photochemotherapyeg. tumours, dysplasia or other growths, non-malignant gynaecologicaldiseases such as menorrhagia, endometriosis and ectopic pregnancy, skindisorders such as psoriasis, actinic keratoses and acne, skin abrasions,and other diseases or infections eg. bacterial, viral or fungalinfections, for example Herpes virus infections. The invention isparticularly suited to the treatment of diseases, disorders orabnormalities where discrete lesions are formed (lesions is used here ina broad sense to include tumours and the like). However, the methods ofthe invention may also be used to treat abnormalities and disorders notcharacterized by a lesion but displaying discrete separable entitieswhich characterize that disease, for example, abnormalities in the bloodor bone marrow indicative of diseases of said blood or marrow orindicative of a disease or disorder located elsewhere in the body whichadditionally may result in the presence of abnormalities in the blood ormarrow, e.g circulating transformed cells.

[0068] The internal and external body surfaces which may be treatedaccording to the invention include the skin and all other epithelial andserosal surfaces, including for example mucosa, the linings of organseg. the respiratory, gastro-intestinal and genito-urinary tracts, andglands with ducts which empty onto such surfaces (e.g. liver, sebaceousglands, mammary glands, salivary glands and seminal vesicles). Inaddition to the skin, such surfaces include for example the lining ofthe vagina, the endometrium and the urothelium. Such surfaces may alsoinclude cavities formed in the body following excision of diseased orcancerous tissue eg. brain cavities following the excision of tumourssuch as gliomas.

[0069] Exemplary surfaces thus include: (i) skin and conjunctiva; (ii)the lining of the mouth, pharynx, oesophagus, stomach, intestines andintestinal appendages, rectum, and anal canal; (iii) the lining of thenasal passages, nasal sinuses, nasopharynx, trachea, bronchi, andbronchioles; (iv) the lining of the ureters, urinary bladder, andurethra; (v) the lining of the vagina, uterine cervix, and uterus; (vi)the parietal and visceral pleura; (vii) the lining of the peritoneal andpelvic cavities, and the surface of the organs contained within thosecavities; (viii) the dura mater and meninges; (ix) any tumours in solidtissues that can be made accessible to photoactivating light e.g. eitherdirectly, at time of surgery, or via an optical fibre inserted through aneedle.

[0070] The compositions of the invention may be formulated inconventional manner optionally with one or more physiologicallyacceptable carriers or excipients, according to techniques well known inthe art. Topical compositions are preferred except when a singlecomposition according to the invention is prepared and a topicalcomposition is not suitable for administration of an agent, e.g.Photofrin® in which case systemic application, at least of that agent,will be necessary. Topical compositions include gels, creams, ointments,sprays, lotions, salves, sticks, soaps, powders, pessaries, aerosols,drops and any of the other conventional pharmaceutical forms in the art.

[0071] Ointments and creams may, for example, be formulated with anaqueous or oily base with the addition of suitable thickening and/orgelling agents. Lotions may be formulated with an aqueous or oily baseand will, in general, also contain one or more emulsifying, dispersing,suspending, thickening or colouring agents. Powders may be formed withthe aid of any suitable powder base. Drops may be formulated with anaqueous or non-aqueous base also comprising one or more dispersing,solubilising or suspending agents. Aerosol sprays are convenientlydelivered from pressurised packs, with the use of a suitable propellant.

[0072] Alternatively, the surface penetration assisting agent is appliedtopically in a separate step, and the vascular stroma-localizingphotosensitizer, e.g. Photofrin® and protoporphyrin precursorphotochemotherapeutic agent, e.g. ALA, optionally together or separatelywith one or more chelating agents may be administered by an alternativeroute e.g. orally or parenterally for example by intradermal,subcutaneous, intraperitoneal or intravenous injection. Alternativepharmaceutical forms thus include plain or coated tablets, capsules,suspensions and solutions containing the active components optionallytogether with one or more inert conventional carriers and/or diluents,e.g. with corn starch, lactose, sucrose, microcrystalline cellulose,magnesium stearate, polyvinylpyrrolidone, citric acid, tartaric acid,water, water/ethanol, water/glycerol, water/sorbitol,water/polyethyleneglycol, propyleneglycol, stearylalcohol,carboxymethylcellulose or fatty substances such as hard fat or suitablemixtures thereof.

[0073] Following administration to the surface or systemicadministration, or both, the area treated is exposed to light to achievethe photo-chemotherapeutic effect. This can generally be in the order ofa few minutes to 96 hours, preferably 15 minutes to 3 hours. The lengthof time before light administration is also dependant on the mode ofadministration, and also the dose and particular agent employed.

[0074] The irradiation will in general be applied at a dose level of 10to 250 Joules/cm² with an intensity of 20-200 mW/cm² when a laser isused or a dose of 10-540 J/cm² with-an intensity of 50-300 mW/cm² when alamp is applied. At 100 Joules/cm², penetration of the radiation isfound to be relatively deep. Irradiation is preferably performed for 5to 30 minutes, preferably for 15 minutes. A single irradiation may beused or alternatively a light split dose in which the light dose isdelivered in two fractions, e.g. a few minutes to a few hours betweenirradiations, may be used.

[0075] The wavelength of light used for irradiation may be selected toachieve a more efficacious photochemotherapeutic effect. Conventionally,when porphyrins are used in photochemotherapy they are irradiated withlight at about the absorption maximum of the porphyrin. Thus, forexample in the case of the prior art use of ALA in photochemotherapy ofskin cancer, wavelengths in the region 350-640 nm, preferably 610-635 nmwere employed. However, by selecting a broad range of wavelengths forirradiation, extending beyond the absorption maximum of the porphyrin,the photosensitizing effect may be enhanced. Whilst not wishing to bebound by theory, this is thought to be due to the fact that when Pp, andother porphyrins, are exposed to light having wavelengths within itsabsorption spectrum, it is degraded into various photo-productsincluding in particular photoprotoporphyrin (PPp). PPp is a chlorin andhas a considerable photo-sensitizing effect; its absorption spectrumstretches out to longer wavelengths beyond the wavelengths at which Ppabsorbs ie. up to almost 700 nm (Pp absorbs almost no light above 650nm). Other agents have been identified for use in PDT which absorb lightof even higher wavelengths. Thus in conventional photochemotherapy, thewavelengths used do not excite PPp and hence do not obtain the benefitof its additional photosensitizing effect. Irradiation with wavelengthsof light in the range 350-900 nm has been found to be particularlyeffective although this depends on the agent which is employed. It isparticularly important to include the wavelengths between 600 and 700nm, especially between 630 and 690 nm, specifically the range 630 to 670nm.

[0076] A further aspect of the invention thus provides a method ofphotochemotherapeutic treatment of disorders or abnormalities ofexternal or internal surfaces of the body, comprising administering tothe affected surfaces, a composition or product as hereinbefore defined,and exposing said surfaces to light, preferably to light in thewavelength region 350-900 nm. Alternatively however a light of a narrowwavelength may be used, e.g. when a laser is used, light at a wavelengtharound 630 nm may be used.

[0077] Methods for irradiation of different areas of the body, eg. bylamps or lasers are well known in the art (see for example Van denBergh, Chemistry in Britain, May 1986 p. 430-439).

[0078] It will be appreciated that the method of therapy using compoundsof the invention inevitably involves the fluorescence of the disorder orabnormality to be treated. Whilst the intensity of this fluorescence maybe used to eliminate abnormal cells, the localization of thefluorescence may be used to visualize the size, extent and situation ofthe abnormality or disorder. This is made possible through the abilityof the agents used in accordance with the invention to preferentiallylocalize to non-normal tissue.

[0079] The abnormality or disorder thus identified or confirmed at thesite of investigation may then be treated through alternativetherapeutic techniques e.g. surgical or chemical treatment, or by themethod of therapy of the invention by continued build up of fluorescenceor through further application of compounds of the invention at theappropriate site. It will be appreciated that diagnostic techniques mayrequire lower levels of fluorescence for visualization than used intherapeutic treatments. Thus, generally, concentration ranges of 1 to50% e.g. 1-5% (w/w) are suitable. Sites, methods and modes ofadministration have been considered before with regard to thetherapeutic uses and are applicable also to diagnostic uses describedhere. The compounds of the invention may also be used for in vitro andin vivo diagnostic techniques, for example for examination of the cellscontained in body fluids. The higher fluorescence associated withnon-normal tissue may conveniently be indicative of an abnormality ordisorder. This method is highly sensitive and may be used for earlydetection of abnormalities or disorders, for example bladder or lungcarcinoma by examination of the epithelial cells in urine or sputumsamples, respectively. Other useful body fluids which may be used fordiagnosis in addition to urine and sputum include blood, semen, tears,stools, spinal fluid etc. Tissue samples or preparations may also beevaluated, for example biopsy tissue or bone marrow samples. The presentinvention thus extends to the use of compounds of the invention, orsalts thereof for diagnosis according to the aforementioned methods forphotochemotherapy, and products and kits for performing said diagnosis.

[0080] A further aspect of the invention relates to a method of in vitrodiagnosis, of abnormalities or disorders by assaying a sample of bodyfluid or tissue of a patient, said method comprising at least thefollowing steps:

[0081] i) admixing said body fluid or tissue with a compound asdescribed hereinbefore,

[0082] ii) exposing said mixture to light,

[0083] iii) ascertaining the level of fluorescence, and

[0084] iv) comparing the level of fluorescence to control levels.

[0085] The invention will now be described in more detail in thefollowing non-limiting Examples, with reference to the drawings inwhich:

[0086]FIG. 1 is a graph showing the averaged results for growth curvesof WiDr human colonic carcinoma transplanted subcutaneously into nudemice given intravenous injections of Photofrin® and/or intraperitonealadministration of ALA, followed, 3 hours later, by laser lightirradiation (632 nm, 150 mW/cm² for 15 min)  Control (no drug, nolight); ▴ Control (light only); ♦ ALA 250 mg/kg, irradiation after 3hours; ▾ Photofrin® 1 mg/kg, irradiation after 3 hours; ▪ ALA 250 mg/kgand Photofrin® 1 mg/kg, irradiation after 3 hours; abscissa shows daysafter treatment; ordinate shows relative tumour volume. Bars indicatedstandard error of mean (SEM) based on at least 3 animals in each group;

[0087]FIG. 2 is a graph showing the averaged results for growth curvesof WiDr human colonic carcinoma transplanted subcutaneously into nudemice given intravenous injections of m-THPC and/or intraperitonealadministration of ALA, followed, 3 hours later, by laser lightirradiation (632 nm, 150 mW/cm² for 15 min).  Control (no drug, nolight); ▴ Control (light only); ♦ ALA 250 mg/kg, irradiation after 3hours; ▾ m-THPC 75 μg/kg, irradiation after 3 hours; ▪ ALA 250 mg/kg andm-THPC 75 μg/kg, irradiation after 3 hours; abscissa shows days aftertreatment; ordinate shows relative tumour volume. Bars indicatedstandard error of mean (SEM) based on at least 3 animals in each group;

[0088]FIG. 3—Fluorescence photomicrographs of human rectal papillaryvillous adenomas from a 75-year old male (A) and an 87-year old female(B), sampled 44 hours after i.v. injection of 2 mg/kg Photofrin® (A) and4.5 hours after oral administration of 60 mg/kg ALA (B).

[0089]FIG. 4 is as FIG. 1 in which intravenous injections of chlorin e6and/or intraperitoneal administrations of ALA are made, followed, 1 hourlater by lamp irradiation. × Control; ▴ ALA 250 mg/kg; ▪ chlorin e6 1mg/kg; ♦ ALA 250 mg/kg and chlorin e6 1 mg/kg. The abscissa shows daysafter treatment; ordinate shows relative tumour volume;

[0090]FIG. 5 is as FIG. 1 in which intravenous injections of A1PcS_(2a)and/or intraperitoneal administrations of 5-ALA methyl ester are made,followed 1 hour later by lamp irradiation. ♦ Control; ▪ A1PcS_(2a) 1mg/kg; ▴ 5-ALA methyl ester 273 mg/kg; × 5-ALA methyl ester 273 mg/kgand A1PcS_(2a) 1 mg/kg. The abscissa shows days after treatment;ordinate shows relative tumour volume;

[0091]FIG. 6 is as FIG. 1 in which intravenous injections of A1PcS₄and/or intraperitoneal administrations of 5-ALA butyl ester are made,followed one hour later by lamp irradiation. ♦ Control; ▪ A1PcS₄ 5mg/kg; × A1PcS₄ 1 mg/kg;

5-ALA butyl ester 338 mg/kg; Δ 5-ALA butyl ester 338 mg/kg and A1PcS₄ 1mg/kg. The abscissa shows days after treatment; ordinate shows relativetumour volume;

[0092]FIG. 7 is as FIG. 1 in which intravenous injections of A1PcS_(2a)and/or intraperitoneal administration of 5-ALA butyl ester are made,followed 1 hour later by lamp irradiation. ▴ Control; ▪ A1PcS_(2a) 1mg/kg; × 5-ALA butyl ester 338 mg/kg; ♦ 5-ALA butyl ester 338 mg/kg andA1PcS_(2a) 1 mg/kg. The abscissa shows days after treatment; ordinateshows relative tumour volume;

[0093]FIG. 8 is as FIG. 1 in which intravenous injections of A1PcS₄and/or intraperitoneal administrations of 5-ALA methyl ester are made,followed one hour later by lamp irradiation. ▪ Control; ▴ A1PcS₄ 1mg/kg; × 5-ALA methyl ester 273 mg/kg; ♦ 5-ALA methyl ester 273 mg/kgand A1PcS₄ 1 mg/kg. The abscissa shows days after treatment; ordinateshows relative tumour volume.

EXAMPLE 1

[0094] Formulations

[0095] 1.1 ALA-containing Cream for Topical Administration

[0096] An ALA-containing cream, containing 5-30% ALA, is prepared byadmixing ALA with a commercially available cream base.

[0097] A 20% ALA cream was prepared by admixture with “Urguentum Merck”cream base (available from Merck) consisting of silicon dioxide,paraffin liq., vaseline, album, cetostearol., polysorbat. 40, glycerolmonostearate, Miglyol®812 (a mixture of plant fatty acids),polypropyleneglycol., and purified water.

[0098] 1.2 ALA for Systemic Administration

[0099] For oral administration, ALA is dissolved in acidic soft drinks.For intravenous administration ALA is dissolved in isotonic saline.

[0100] 1.3 Photofrin® for systemic administration

[0101] Photofrin® is dissolved in 5% glucose solution.

EXAMPLE 2

[0102] PDT using ALA+Photofrin®

[0103] Materials and Methods

[0104] Chemicals

[0105] 5-aminolevulinic acid (ALA) hydrochloride was purchased fromSigma Chemical Company (St. Louis, Mo.). ALA was freshly dissolved inisotonic saline and given intraperitoneally to mice. Photofrin® wasobtained from Quadra Logic Technologies (Vancouver, Canada). Thesolution of Photofrin® was made up in isotonic solution containing 5%dextrose and given to mice intravenously via the tail vein.

[0106] Animals and Tumor Line

[0107] Female Balb/c nu/nu nude mice were obtained from The AnimalDepartment, The Norwegian Radium Hospital, housed 10 per cage and keptunder specific-pathogen-free conditions. The mice were 6 weeks old andweighed 20-22 g when the experiments started. The WiDr human coloniccarcinoma, used in the present study, was propagated by serialtransplantation into the nude mice. Non-necrotic tumor material forinoculation was obtained by sterile dissection of large flange tumorsfrom syngeneic mice. Macroscopically viable tumor tissue was gentlyminced with a pair of scissors and forced repeatedly through sterileneedles of diminishing sizes from 19-gauge to 25-gauge to make atumor-tissue suspension, 0.02 ml of which was then injected into thedorsal side of the right hind foot of each mouse. The rate of successfultransplantations was nearly 100% in the present experiments. Nospontaneous necrosis was observed in the tumors which grew to reach 5-7mm (about 14 days after inoculation) transverse diameter on the day oftreatment, as measured with calipers every second day. The tumor volumewas calculated using the following formula:

V=π/6(D ₁ ×D ₂ ×D ₃)

[0108] where D₁, D₂ and D₃ are three orthogonal diameters of the tumorswhich were measured daily by a caliper.

[0109] Light Exposure

[0110] Unanesthetized mice were fixed in Lucite jigs specially designedfor irradiation. The tumor area was exposed to red light from adicyanomethylane-2-methyl-6-(p-dimethylaminostyryl)-4 H-pyran (DCM) dyelaser pumped by a 5W argon ion laser (Spectra Physics, 164). The tuningrange was 610-690 nm. The dye laser was tuned at 632 for bothALA-derived PpIX and Photofrin®, the tuning being controlled by means ofa monochromator. The laser beam was defocused by means of a microscopicocular. The light was delivered at a fluence rate of 150 mW/cm² for15-min exposure. The fluence rate of the light on the tumor area wasregularly controlled by a calibrated integrating sphere with aphotodiode coupled to a digital multimeter (Keithley Instruments,Germany) before and immediately after light illumination.

[0111] PDT Efficiency Using ALA or Photofrin® Alone, or a Combination ofALA with Photofrin®

[0112] Mice with tumors of the appropriate size were divided into 5groups (at least 3 animals for each group): group 1 (controls), micewere given neither ALA, Photofrin® nor light, only intraperitonealadministration of 0.1 ml saline; group 2 (control-light only) thetumours were irradiated at the same doses as those for groups receivingPDT treatment; group 3 (ALA alone), mice were given an intraperitonealinjection of ALA of 250 mg/kg body weight, followed, 3 hours later, bylight exposure as described above; group 4 (Photofrin® alone), mice weregiven an intravenous injection of Photofrin® of 1 mg/kg body weight,followed, 3 hours later, by light irradiation; group 5 (ALA andPhotofrin®), mice were given an intraperitoneal injection of 250 mg/kgALA and an intravenous injection of 1 mg/kg Photofrin®, the tumors wereexposed to light 3 hours for both-ALA and Photofrin®. Responses of thetreated tumors were evaluated as tumor regression/regrowth time. Thesize of the tumors were measured every day and when the treated tumorsreached a volume 5 times that of the volume on the day just before lightirradiation, the mice were sacrificed. The data based on themeasurements on tumor volumes from each group were pooled to representmean tumor growth curves.

[0113] Results

[0114] The growth of the tumors exposed to light 3 hours after anintraperitoneal injection of ALA or an intravenous administration ofPhotofrin® alone or both ALA and Photofrin® is shown in FIG. 1. Thecontrol tumors (neither drug nor light) grew exponentially with adoubling time of about 5 days. Laser light given to tumors of micereceiving ALA had an effect on the tumor growth. No effect was seenafter PDT with Photofrin® alone at a dose of 1 mg/kg, a dose that doesnot induce any skin phototoxicity (data not shown). PDT with acombination of ALA (250 mg/kg) and Photofrin® (1 mg/kg) inhibited thegrowth of the tumors more efficiently than did PDT using ALA (250 mg/kg)alone.

EXAMPLE 3

[0115] PDT using ALA+m-THPC

[0116] PDT was performed essentially as described in Example 2 using thefollowing groups of animals, with at least 3 animals per group: group 1(control), mice were given neither ALA (m-THPC) nor light, onlyintraperitoneal administration of 0.1 ml saline; group 2 (light only),tumors were irradiated with light at the same doses as those for groupsof PDT treatment; group 3 (ALA alone), mice were given anintraperitoneal injection of ALA of 250 mg/kg body weight, followed, 3hours later, by light exposure (632 nm) as described earlier; group 4(m-THPC alone), mice were given an intravenous injection of m-THPC of 75μg/kg body weight (a dose that does not induce any skin phototoxicity),followed, 3 hours later, by light irradiation (652 nm); group 5 (ALA andm-THPC), mice were given an intraperitoneal injection of 250 mg/kg ALAand an intravenous injection of 75 μg/kg m-THPC, the tumours wereexposed to light (at respective wavelengths) 3 hours for both ALA andm-THPC. Responses of the treated tumors were evaluated as describedpreviously.

[0117] Results

[0118]FIG. 2 shows that the control tumors (neither drug nor light) grewexponentially with a doubling time of about 5 days. Laser light given totumors of mice receiving only ALA had an effect on the tumor growth, butno effect was seen after PDT with m-THPC at a dose of 75 μg/kg. PDT witha combination of ALA (250 mg/kg) and m-THPC (75 μg/kg) synergisticallyenhanced the effect on inhibiting the tumor growth.

EXAMPLE 4

[0119] Distribution of ALA and Photofrin®

[0120] Methods

[0121] Human rectal papillary villous adenomas from 2 patients withsevere dysplasia and with a diarrheal history for some months beforediagnosis were sampled 44 hours after intravenous injection of 2 mg/kgbody weight Photofrin® or 4.5 hours after oral administration of 60mg/kg ALA. The samples were immediately immersed in liquid nitrogen,then mounted in medium (Tissue Tek II embedding compound: BDH, Poole,UK). Frozen tissue sections were cut with a cryostat to a thickness of 8Am and mounted on clean glass slides. The fluorescence localizationpatterns of ALA-induced PpIX and Photofrin were studied by fluorescencemicroscopy. The fluorescence microscopy was carried out with an Axioplanmicroscope (Zeiss, Germany). The filter combination comprised a 390-440nm excitation filter, a 460 nm beam splitter and a >600 nm emissionfilter. The fluorescence images were recorded by a CCD camera (AstromedCCD 3200, Cambridge, UK) and an image processing unit (Astromed/Visilog,PC 486DX2 66 MHz VL).

[0122] Results

[0123] The results are shown in FIG. 3 for the localization ofPhotofrin® (A) and ALA (B). The adenoma in (A) was from a male patientaged 75, and in (B), a female patient aged 87. Fluorescence ofPhotofrin® is mainly distributed in the stroma of the tumor tissue,whereas the fluorescence of ALA-induced prophyrins is almost entirelylocalized within the tumor cells.

EXAMPLE 5

[0124] Materials and Methods

[0125] Chemicals

[0126] 5-ALA, 5-ALA methyl ester and 5-ALA butyl ester were manufacturedby Norsk Hydro Research Center, Porsgrunn, Norway.

[0127] 5-ALA (ALA) and ALA-Methyl ester (ME) were dissolved in isotonicsaline to a final concentration of 0.375 mM.

[0128] ALA-Butyl ester (BU) was dissolved in a small amouont of ethanoland diluted further in isotonic saline to a final concentration of 0.375mM (final ethanol concentration was 2% v/v).

[0129] Aluminium phthalocyanine di-sulfonate (A1PcS_(2a)) (PorphyrinProducts Inc.) dissolved in a few drops of 1M NaOH and diluted inphosphate buffered saline (PBS, 10 MM Na-phosphate pH 7.4/150 mM NaCl)to a final concentration of 0.25 mg/ml.

[0130] Aluminium phthalocyanine tetra-sulfonate (A1PcS₄) (PorphyrinProducts Inc.) dissolved in PBS to a final concentration of 0.25 g/ml,or to 1.25 mg/ml for the high dose experiment in Example 5.3.

[0131] Photofrin (PII) (Quadra Logic Technologies) was dissolved in 5%glucose in H₂O to a final concentration of 0.25 mg/ml.

[0132] Chlorin e6 (e6)(Porphyrin Products Inc.) was dissolved in PBS toa final concentration of 0.25 mg/ml.

[0133] ALA, ALA methyl ester or ALA butyl ester were administeredintraperitoneally (i.p.), whereas the sensitizers were injectedintravenously (i.v.).

[0134] Animals

[0135] The animals used were as described in Example 2.

[0136] All animals received the same amount of ALA (1.5 mmole), eitheras the free acid or in the form of an ester. Due to differences in themolecular weights between ALA and the esters, the animals received 250mg/kg ALA, 278 mg/kg ALA methyl ester and 338 mg/kg of ALA butyl ester.

[0137] Experimental

[0138] Suspensions of the human tumour (Colon carcinoma WiDr-propagatedby serial transplantation) was prepared from non-necrotic areas of therespective tumours and injected (20 μl) into the right hind foot of eachmouse. When the tumors have reached a diameter of 5-7 mm, each mouse wasinjected with the drugs and controls as specified, in Examples 5.1through 5.5. Injection volume: 100 μl per mouse (approx. 25 gbodyweight).

[0139] Illumination occurred one hour after injection of the drugsinstead of three hours that was used in previous examples. In contrastto previous examples, a broad-band lamp that covers the range of 600 to700 nm (Curelight, patent applied for by PhotoCure AS) was used insteadof the laser. This was because the light should cover combinations ofphthalocyanines (absorption maximum 670 nm) and protoporphyrin IX(absorption maximum 630 nm) induced by ALA or ALA-esters, respectively.

[0140] However, the lamp produces light with a lower intensity than thelaser. Thus, combinations of ALA and Photofrin that were effective inthe previous examples when the laser was used will no longer beeffective when the lamp is used. This illumination time is optimal forobtaining a vascular effect for most sensitizers and optimal for theesters of ALA but sub-optimal for ALA.

[0141] The average tumour volume in each group (mean ±SD) was calculatedand plotted against time. The experiment was terminated when the tumorvolume had reached 4-5 times the initial volume.

EXAMPLE 5.1 ALA with chlorin e6

[0142] Mice with tumours of the appropriate size were divided into threegroups of 4-5 mice. Group 1: Control (100 μl physiological saline i.p.)Group 2: 5-ALA 250 mg/kg (1.5 mmole) i.p. + Chlorin e6 (1 mg/kg) i.v.Group 3: 5-ALA 250 mg/kg (1.5 mmole) i.p. Group 4: Chlorin e6 (1 mg/kg)i.v.

[0143] One hour after injection of the drugs the mice were irradiatedusing the Curelight broad-band lamp (161 mW/cm² for 15 minutes-144.9J/cm²).

[0144] Responses of the tumors were evaluated as regression/regrowthtime. The size of the tumours were measured every second day and thetumour volumes calculated according to the formula in Example 2. Themice were sacrificed when the tumour volume had reached 5 times theinitial volume. For each time point, the mean (and the standarddeviation of the mean) tumour volume (n=4-5) were calculated. The datawere then submitted to statistical analysis (Q-test/90% confidenceinterval) and extreme values were rejected. The standard deviations werein the majority of cases ≦1.

[0145] Results

[0146] The results are shown in FIG. 4. Standard deviation bars havebeen omitted for clarity. It can be seen from the figure that thecontrol tumors reached 4× initial volume within 10 days, and that thecontrol tumors displayed a logarithmic growth. Furthermore, ALA andchlorin e6 when used alone had no effect at the doses used. However, thecombination of ALA and chlorin e6 delayed tumor growth significantly. Infact, it took 39 days for the tumor that had been treated with thecombination to reach 4 times the initial volume.

EXAMPLE 5.2 ALA methyl ester with A1PcS_(2a)

[0147] Mice with tumours of the appropriate size were divided into threegroups of 4-5 mice. Group 1: Control (100 μl physiological saline i.p.)Group 2: A1PcS_(2a) (1 mg/kg) i.v. Group 3: ALA-methyl ester (273 mg/kg)(1.5 mmole) i.p. Group 4: ALA-methyl ester (273 mg/kg) (1.5 mmole)i.p. + A1PcS_(2a) (1 mg/kg) i.v.

[0148] Mice were irradiated one hour after injection and responses oftumours were evaluated as regression/regrowth time according to Example5.1.

[0149] Results

[0150] The results are shown in FIG. 5. It can be seen from the figurethat the control tumours reached 4× initial volume in 10 days, and thatthe control tumours displayed logarithmic growth. ALA-methyl ester hadno anti-tumour effect at the dose used, whereas the A1PcS_(2a) displayeda slight effect. Surprisingly, the combination (ALA methylester+A1PcS_(2a)) resulted in a massive effect. In fact, the tumourvolumes did not increase significantly during as long as 40 days aftertreatment.

EXAMPLE 5.3 ALA Butyl ester with A1PcS₄

[0151] Mice with tumors of the appropriate size were divided into fivegroups of 4-5 mice. Group 1: Control (100 μl 2% ethanol i.p.) Group 2:5-ALA Butyl-ester 338 mg/kg (1.5 mmole) i.p. + A1PcS₄ (1 mg/kg) i.v.Group 3: A1PcS₄ (5 mg/kg) i.v. Group 4: A1PcS₄ (1 mg/kg) i.v. Group 5:5-ALA Butyl-ester 338 mg/kg (1.5 mmole) i.p.

[0152] The ethanol was used as the control treatment since the ALA butylester formulation contained approx. 2% of ethanol. Mice were irradiatedone hour after injection and responses of the tumours were evaluated asregression/regrowth time according to Example 5.1.

[0153] Results

[0154] The results are shown in FIG. 6. It can be seen from the figurethat the control tumours (2% ethanol) and the tumors that were treatedwith ALA butyl ester reached 4 times the initial volume in 9 and 11days, respectively. It is also seen that the control tumours grewlogarithmically. A1PcS₄ (1 mg/kg) is seen to have a moderate effect ontumour growth. However, the tumours that were treated with thecombination of ALA butyl ester and A1PcS₄ showed a strongly delayedgrowth, almost identical to that obtained with the high dose of A1PcS₄(5 mg/kg). However, the use of the high dose A1PcS₄ resulted in adevelopment of a large oedema. By use of the combination, theanti-tumour effect was the same as for the high dose A1PcS₄, whereas theinitial oedema was strongly reduced.

EXAMPLE 5.4 ALA Butyl Ester with A1PcS_(2a)

[0155] Mice with tumours of the appropriate size were divided into threegroups of 4-5 mice. Group 1: Control (100 μl physiological saline i.p.)Group 2: 5-ALA Butyl-ester 338 mg/kg (1.5 mmole) i.p. + A1PcS₂ (1 mg/kg)i.v. Group 3: A1PcS₂ (1 mg/kg) i.v. Group 4: 5-ALA Butyl-ester 338 mg/kg(1.5 mmole) i.p.

[0156] The mice were irradiated one hour after injection and responsesof tumours were evaluated as regression/regrowth time according toExample 5.1.

[0157] Results

[0158] The results are shown in FIG. 7. It can be seen from the figurethat the control tumours reached 4× initial volume in 10 days, and thatthe tumours grew logarithmically. As seen before, the butyl ester hadalmost no effect on the tumours, whereas the AlPcS_(2a) had an immediateeffect (4× volume within 14 days). Again the combination significantlydelayed tumour growth, resulting in a slow regrowth; it is seen (afterextrapolation) that 4× initial volume will be reached in approximately30 days.

EXAMPLE 5.5 ALA Methyl Ester with A1PcS₄

[0159] Mice with tumours of the appropriate size were divided into threegroups of 4-5 mice. Group 1: Control (100 μl physiological saline i.p.)Group 2: 5-ALA methyl ester 273 mg/kg (1.5 mmole) i.p. + A1PcS₄ (1mg/kg) i.v. Group 3: 5-ALA methyl ester 273 mg/kg (1.5 mmole) i.p. Group4: A1PcS₄ (1 mg/kg) i.v.

[0160] Mice were irradiated one hour after injection and responses ofthe tumours were evaluated as regression/regrowth time according toExample 5.1.

[0161] Results

[0162] The results are shown in FIG. 8. It can be seen from the figurethat the control tumours reached 4 times the initial volume in 10 days,and the growth of the control tumours occurred in a logarithmic manner.As seen before, the methyl ester did not have any effect on tumorgrowth, whereas the A1PcS₄ had an intermediate effect. Strikingly, thecombination of the ALA methyl ester+A1PcS₄ resulted initially in asubstantial reduction of tumour volume followed by a slow regrowth ofthe tumour. In fact, this is the only combination that actually resultedin an initial loss of tumour size. The tumour reached 4 times theinitial volume at approx. 35 days.

1. A pharmaceutical composition comprising a protoporphyrin precursorphotochemotherapeutic agent together with a vascular stroma-localizingphotosensitizers, optionally together with at least one surfacepenetration assisting agent and optionally with one or more chelatingagents.
 2. A pharmaceutical composition as defined in claim 1 for thetreatment of disorders or abnormalities of external or internal surfacesof the body which are responsive to photochemotherapy.
 3. Apharmaceutical composition as claimed in claim 2 wherein the therapeuticefficacy is enhanced relative to the use of the photochemotherapeuticagent or the photosensitizer alone.
 4. A pharmaceutical composition asclaimed in claim 2 or 3 wherein the therapeutic efficacy issynergistically enhanced.
 5. A pharmaceutical composition as claimed inany one of claims 1 to 4 wherein the vascular stroma-localizingphotosensitizer is provided at a sub-therapeutic dose.
 6. Apharmaceutical composition as claimed in any one of claims 1 to 5wherein the vascular stroma-localizing photosensitizer is aHematoporphyrin, or a chlorin or a sulphonated phthalocyanine, or aprecursor or derivative thereof.
 7. A pharmaceutical composition asclaimed in claim 6 wherein the vascular stroma-localizing agent isPhotofrin®, m-THPC, chlorin e6, aluminium phthalocyanine di-sulfonate oraluminium phthalocyanine tetra-sulfonate, or a precursor or derivativethereof.
 8. A pharmaceutical composition as claimed in any one of claims1 to 7 wherein the protoporphyrin precursor is ALA or a precursor orderivative thereof.
 9. A pharmaceutical composition as claimed in claim8 where the ALA derivative is an ALA ester.
 10. A pharmaceuticalcomposition as claimed in any one of claims 1 to 9 wherein thesurface-penetration assisting agent is DMSO.
 11. A pharmaceuticalcomposition as claimed in any one of claims 1 to 10 comprising ALA or aprecursor or derivative thereof, Photofrin®, DMSO, EDTA anddesferrioxamine.
 12. The use of a protoporphyrin precursorphotochemotherapeutic agent together with a vascular stroma-localizingphotosensitizer, optionally together with at least one surfacepenetration assisting agent and optionally with one or more chelatingagents, as defined in any one of claims 1 to 11 in the preparation of-acomposition for the treatment of disorders or abnormalities of externalor internal surfaces of the body which are responsive tophotochemotherapy.
 13. A product comprising a protoporphyrin precursorphotochemotherapeutic agent and a vascular stroma-localizingphotosensitizer, optionally together with at least onesurface-penetration assisting agent, and optionally one or morechelating agents, as defined in any one of claims 1 to 11, as a combinedpreparation for simultaneous, separate or sequential use in treatingdisorders or abnormalities of external or internal surfaces of the bodywhich are responsive to photochemotherapy.
 14. The use of aprotoporphyrin precursor photochemotherapeutic agent and a vascularstroma-localizing photosensitizer, optionally together with at least onesurface-penetration assisting agent, and optionally one or morechelating agents, as defined in any one of claims 1 to 11, in thepreparation of a product for simultaneous, separate or sequential use intreatment of disorders or abnormalities of external or internal surfacesof the body which are responsive to photochemotherapy.
 15. Apharmaceutical composition, product or use and claimed in any one ofclaims 2 to 14 wherein the total dosage of the vascularstroma-localizing photosensitizer administered is in the range of 0.01to 10 mg/kg body weight and for the protoporphyrin precursorphotochemotherapeutic agent is in the range of 1 to 500 mg/kg bodyweight.
 16. A pharmaceutical composition, product or use as claimed inany one of claims 2 to 15 wherein photochemotherapy is performed byirradiation with wavelengths of light in the range 350-900 nm.
 17. A kitfor use in photochemotherapy of disorders or abnormalities of externalor internal surfaces of the body comprising: a) a first containercontaining a protoporphyrin precursor photochemotherapeutic agent, asdefined in any one of claims 2 to 4, 8 or 9; b) a second containercontaining a vascular stroma-localizing photosensitizer, as defined inany one of claims 2 to 7; and optionally c) at least onesurface-penetrating agent contained within said first or secondcontainer or in a third container as defined in any one of claims 2 or10; and/or d) one or more chelating agents contained either within saidfirst, second or third container or in a fourth container; wherein saidfirst or second container may be absent and the agent or photosensitizerof a) or b) above is present in one of the other containers present inthe kit.
 18. A method of photochemotherapeutic treatment of disorders orabnormalities of external or internal surfaces of the body, comprisingadministering to the affected surfaces, a pharmaceutical composition orproduct as defined in any one of claims 1 to 11, 13 or 15, and exposingsaid surfaces to light, preferably to light in the wavelength region350-900 nm.
 19. A method of in vitro diagnosis of abnormalities ordisorders by assaying a sample of body fluid or tissue of a patient,said method comprising at least the following steps: i) admixing saidbody fluid or tissue with a pharmaceutical composition as defined in anyone of claims 1 to 11, ii) exposing said mixture to light, iii)ascertaining the level of fluorescence, and iv) comparing the level offluorescence to control levels.